The long term objective of our research is the elucidation of the molecular mechanism of the acute and chronic effects of opiate narcotic analgesics. Our approach to this problem rests primarily on the study of the endogenous opioid peptide-opiate receptor system. A knowledge of the workings of this system would have wide-ranging implications and, we feel, will lead towards an understanding of the actions of exogenous opiates. Our focus will be on opiate receptors, their properties and isolation. The proposed research can be divided into 4 categories: 1) membrane-bound opiate receptors 2) soluble opiate receptors 3) opiate receptors and endogenous opioids in tissue culture and 4) involvement of the endogenous opioid system in the control of pain and aversion. In studies with membrane-bound receptors we will attempt further characterization of agonist and antagonist binding and of receptor subtypes by thermodynamic and kinetic experiments and selective inhibition or inactivation of receptor subclasses. In collaboration with its developers, Drs. Hirth and Goeldner, a novel method for covalent labeling called "energy transfer photoaffinity labeling" will be applied to opiate receptors. Good yields of solubilized opiate receptors have been obtained by us from nonmammalian and mammalian brain. We plan to characterize these by physical chemical methods and by reconstitution into liposomes and cell membranes. Purification of solubilized receptors and separation of receptor subtypes will be carried out. Attempts will be made to raise monoclonal antibodies, which would aid in further purification and make feasible molecular biological experiments. Tissue culture of spinal cord with attached dorsal root ganglia from fetal mice will be used for studies of opiate receptor subtypes and opioid peptides in tolerance and under various environmental conditions. Fluorescent enkephalin derivatives will be used in video-intensification fluorescence microscopic experiments to learn about receptor distribution and internalization. An approach begun about 2 years ago in this laboratory is the study of the role of endogenous opioids and opiate receptors in the regulation of pain and aversion, using behavioral and brain stimulation procedures. The role of opiate receptor subclasses and endogenous opioids in the modulation of pain responses organized at different levels of the CNS will be assessed.